ADP-ribosylation factors (ARFs) are multi-functional, multi-domain, proteins that regulate vesicular trafficking in the ER and Golgi (and elsewhere) by associating with a lipid membrane after being activated by binding GTP. This facilitates ARF interaction with effectors [e.g., the coat protein beta-COP found on type I Golgi-derived vesicles, the G-protein betagamma, or a specific isoform of phospholipase D (PLD)] and to recruit to membranes proteins that cause budding and vesicle formation. ARFs were first identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity. ARL proteins are very similar in structure to ARFs, but were initially believed not to activate cholera toxin and were, therefore, named ARF-like (ARL). After localizing ARL1 to the Golgi in Saccharomyces cerevisiae, consistent with a role in vesicular trafficking, and finding evidence of PLD activation by recombinant rARL1, we compared ARF and ARL function more systematically. GTPgammaS binding by rARL1 and rARF1 was influenced dramatically and differently by phospholipids and detergents, as was their activation of PLD and cholera toxin. Activation of both by rARL1 was unequivocal, albeit less than ARF activation. Comparison of deduced amino acid sequences of human ARL1 with those of four other human ARLs and five human ARFs revealed the greatest identity (58%) with class II ARFs. (It is 57% identical to human ARF1 and we had earlier defined functional consequences of some of those differences.) ARLs 2 and 3 failed to activate PLD under conditions effective for ARL1. We conclude that ARL1 is part of a continuum of ARF and ARL proteins that are similar in structure and probably function, but differ in ways that are physiologically important, as evidenced by the evolutionary persistence of what seem, in many instances, to be rather subtle structural differences.